Trent C et al. (1981) International C. elegans Meeting "mutants that affect egg-laying in C elegans."

Horvitz HR, Trent C, Tsung N

[International C. elegans Meeting, 1981]

Marie L McGovern et al. (2005) International Worm Meeting "EGL-32 Functions in Sperm to Regulate Egg-laying in C. elegans"

Ling Yu, Marie L McGovern, Cathy Savage-Dunn

[International Worm Meeting, 2005]

We have uncovered a signal in sperm that regulates egg-laying in C.elegans. Hermaphrodites integrate many internal and external cues in deciding whether or not to actively lay eggs. One well known external cue is determining the presence or absence of food. We have discovered an internal cue, the presence of sperm, which is also involved in egg-laying. C.elegans are either hermaphroditic or males. The hermaphrodites do not produce sperm and oocytes at the same time. In a hermaphrodite sperm are exclusively produced during the fourth larval stage. When the worms undergo their final molt to become adults they switch to producing exclusively oocytes and will do so for the remainder of their life. Males produce exclusively sperm for their entire lives. Mutant egl-32 hermaphrodites retain about twice as many eggs as wild-type hermaphrodites. When wild-type sperm is introduced into mutant egl-32 hermaphrodites via mating, the worms begin to retain fewer eggs. When the inverse experiment is performed egl-32 males mated with wild-type hermaphrodite), the wild-type hermaphrodites begin to retain more eggs. These results suggest that sperm regulate egg-laying in C.elegans.

Trent C et al. (1983) International C. elegans Meeting "egg-laying defective mutants of C elegans."

Trent C, Tsung N, Horvitz HR, Desai C

[International C. elegans Meeting, 1983]

Tian, C. et al. (2009) International Worm Meeting "SEM-2 is an SRY-box transcription factor required for egg laying in C. elegans."

Stern, M., Shi, H., Tian, C., Liu, J., Colledge, C., Waterston, R.

[International Worm Meeting, 2009]

C. elegans egg laying system includes the vulva, the gonad, the hermaphrodite specific neurons and the egg-laying muscles. sem-2(n1343) mutant animals lack all the egg-laying vulval and uterine muscles (collectively called sex muscles) and are therefore egg-laying defective. The sex muscles are derived from two sex myoblasts (SMs), which are descendants of the M cell, the progenitor of the postembryonic mesodermal lineage. The absence of the sex muscles in n1343 mutants is due to a fate transformation of the SMs to their sister cells, the bodywall muscles (BWMs). We have found that sem-2(n1343) is a mutation in sox-1(C32E12.5) based on results from cosmid rescue and RNAi knockdown experiments. Furthermore, the n1343 allele contains a TC1 transposon insertion in the first 4.5kb intron of sem-2. In order to determine how sem-2 functions in regulating the SM/BWM fate decision, we examined sem-2 expression using a functional GFP::SEM-2 fusion construct. SEM-2::GFP is present in the nuclei of the SM mother cells (M. vlpa and M. vrpa), SM cells and their descendants as well as a variety of other cell types, including hypodermal cells and gut cells. By performing genetic and molecular epistasis experiments, we found that sem-2 expression in the M lineage is under the control of both dorsal-ventral and anterior-posterior patterning mechanisms that include the LIN-12/Notch pathway, the SMA-9/TGF-beta pathway and the Wnt/beta-catenin asymmetry pathway. We further found that the expression of sem-2 in the M lineage requires a conserved Hox/PBC binding site in its first intron and that this site is disrupted by the TC1 insertion in n1343 mutants. Previous studies have shown that the Hox factors MAB-5 and LIN-39 and the PBC Hox cofactor CEH-20 all function in the M lineage. We are currently testing the hypothesis that sem-2 is a direct target of Hox/PBC in the M lineage.

Dong M-Q et al. (1998) East Coast Worm Meeting "Three G Protein Inhibitors Stimulate Egg Laying in C. elegans"

Koelle MR, Dong M-Q

[East Coast Worm Meeting, 1998]

RGS proteins (regulators of G protein signaling) inhibit G proteins signaling by directly converting active G proteins (GTP-bound) into their inactive GDP-bound forms. Eleven genes with RGS homology have been identified in the C. elegans genome. We found that only three of them, egl-10, C05B5.7, and F16H9.1, stimulate egg laying when they are overexpressed, suggesting that these three genes may somehow function together to control egg laying. EGL-10 has previously been shown to inhibit GOA-1, a G protein alpha subunit, which in turn inhibits egg laying1. Our current interest is in understanding the roles of C05B5.7 and F16H9.1 in the regulation of egg laying, and in understanding the relationship among the three RGS genes and goa-1. We have obtained cDNAs for C05B5.7 and F16H9.1, which encode the most similar pair of RGS proteins in C. elegans (77% identity in the RGS domain and 64% identity overall). We examined the expression patterns of C05B5.7 and F16H9.1 by injecting promoter::GFP constructs into worms. Expression of C05B5.7::GFP was found in most or all neurons, resembling the expression patterns of both egl-10 and goa-1. F16H9.1::GFP was expressed in a large subset of neurons and in the uterine muscles. We are analyzing a C05B5.7 knock-out strain obtained for us by NemaPharm. So far we have not found any egg-laying defects in the C05B5.7 single mutant. However, preliminary data suggest that the C05B5.7 mutation enhances the egg-laying defect of n480, a weak egl-10 allele. We also found that overexpression of C05B5.7 from an integrated transgene array rescued the egg-laying defect of an egl-10 null mutant. We are trying to obtain F16H9.1 mutants to complete our studies. As C05B5.7 and F16H9.1 may be functionally redundant, given the close similarity in their protein sequences and expression patterns, we may need to knock out both genes to understand their functions. Biochemical analysis of the three RGS proteins are in progress. Our short-term goal is to examine whether and how well C05B5.7 and F16H9.1 proteins stimulate the GTPase activity of GOA-1 in vitro as compared to EGL-10. This may help explain why there are multiple RGS genes involved in the signaling pathway that regulates egg laying. 1. Koelle, M.R. and Horvitz, H.R. (1996) Cell 84, 115-125

Ludewig, Andreas H. et al. (2013) International Worm Meeting "Low population density increases lifespan and delays egg laying of C. elegans hermaphrodites."

Doering, Frank, Schroeder, Frank C., Ludewig, Andreas H.

[International Worm Meeting, 2013]

In C. elegans, high population density (overcrowding) has been shown to trigger developmental arrest and dauer formation within early larval stages (Golden and Riddle 1982). The actual population density signal sensed by the worms includes a synergistic mixture of ascaroside pheromones as well as small signalling peptides (Ludewig and Schroeder, 2013; Yamada et al., 2010). However, many aspects of population density-dependent signaling events remain poorly understood. Here, we compared low and intermediate dense C. elegans populations grown under ad libidum feeding conditions. We found that the number of worms per plate strictly determined their average lifespan whereat the number of worms was inverse correlated to mean lifespan. Isolated worms lived around 4 (!) days longer than animals grown up in larger groups. In 1999, Gems and Riddle demonstrated strong dependence of lifespan on population density for males but not for hermaphrodites. However, in those assays worms were placed on experimental plates as L4 larvae, whereas our assays started with eggs, hinting on some defining events during developmental stages L1-L3 in hermaphrodites. We also found that the time point of first egg lay was correlated with the number of worms per plate. Worms that grew up in isolation laid their first egg 9-11 hours later than worms grown up in groups of 25 or more animals per plate. The overall time course of egg laying from isolated worms was shifted towards later time points and the total number of eggs was reduced by 45%. This population dependent early egg laying (= eel) phenotype is completely abolished in daf-16(mu86) mutants and partially abolished in daf-12(rh61; rh411) mutants. Unexpectedly, we also found precocious first egg laying under mild dietary restriction (DR). Evidence from previous studies suggests that small-molecule signalling may trigger DR early egg laying and population dependent longevity (Ludewig and Schroeder, 2013; Yamada et al., 2010; Yzraelit, 2012). To test this hypothesis, we currently examine candidate small molecules for their influence on those phenomena.

C M Loer et al. (2005) International Worm Meeting "Egg laying behavior in C. elegans social vs. solitary strains"

C M Loer, M Maxwell, M Ailion, K Kent

[International Worm Meeting, 2005]

Some wild isolates of C. elegans feed together ("social" strains) whereas "solitary" strains - including the standard wildtype N2 - do not1,2. A single amino acid difference in the NPR-1 neuropeptide receptor mediates this profound behavioral difference; reduction or loss-of-function results in social behavior 2. Social strains in the lab not only clump together, but also "border," spending most of their time in the thicker edge of a bacterial lawn. Solitary strains engage in much less bordering. Bordering is at least partly a consequence of social strains' preference for the lower oxygen concentration of the lawn edge3. We are currently investigating whether social strains display other behaviors in association with clumping and bordering. In particular, we predicted that social strains might display specific egg laying behaviors that would be advantageous in association with social behaviors. The clumping behavior of social worms results in high local density of conspecifics, which likely results in intense competition for food between individual worms. We investigated whether social adults might compensate by laying eggs away from the border of the lawn where the worms aggregate. We allowed individual young adult worms to lay eggs for 4 hours, and then examined the spatial distribution of eggs. Under 21% O2, both wild isolate social strains (RC301, CB4856) and npr-1 mutants with an N2 background (DA650, IM222) lay >90% of eggs at the border whereas N2 lays 48% of eggs at the border. Therefore, social strains do not disperse eggs, but lay them at the border, where they spend most of their time. Low O2 (7%) reduces the percentage of border eggs in social strains to that of N2 (53%). We have also confirmed that wild social strains lay eggs at a earlier stage of development, and retain fewer eggs in the uterus, whereas npr-1 mutants with an N2 genetic background do not4. For example, 92% of eggs laid by the Hawaiian social strain CB4856 are at early gastrulation or younger versus 8% for N2. As a young adult, CB4856 averages about 5 eggs in the uterus whereas N2 averages about 18 eggs. The mutant npr-1(ad609) is like N2, laying only 4% early eggs and carrying on average 13 eggs in the uterus. Preliminary genetics suggests that the early egg laying trait is semi-dominant and located on LG X. How the suite of behavioral traits associated with social behavior affect individual reproductive success and population dynamics warrants further investigation. Refs: 1Hodgkin & Doniach (1997) Genetics 146:149. 2de Bono & Bargmann (1998) Cell 94:679. 3Gray et al. (2004) Nature 430:317. 4Ailion & Thomas (2001) WBG 17(1):39.

Jung, S.K. et al. (2013) International Worm Meeting "Development of a comprehensive image analysis software package for the analysis of lifespan, locomotion, body length, and egg laying of C. elegans."

Zhong, W., Aleman-Meza, B., Riepe, C.M., Jung, S.K.

[International Worm Meeting, 2013]

It is time-consuming to manually quantify C. elegans phenotypes in lifespan, locomotion, body size, and egg laying We developed an automated image acquisition and analysis system to quantify multiple C. elegans phenotypes. The imaging system is composed of a microscope equipped with a digital camera, a motorized stage, and image analysis software. The image analysis software package contains Lifespan Assay, Locomotion Assay, WormLength Assay, and Egg Counter software. The Lifespan Assay software counts the number of moving worms using two time-lapse images. Inspired by the Parallel Worm Tracker developed by the Goodman lab, the Locomotion Assay software conducts fully automated video analysis and computes the velocity of moving worms. The WormLength Assay and Egg counter software have been developed for body size measurement and automated egg counting, respectively. This software suite can run on any operating system since they were written in Java. We evaluate the performance of the software by various benchmarks. We also demonstrate the application of the software in a pilot chemical screen.

Leung, Angela Ching-Yee et al. (2021) International Worm Meeting "Stimulation of egg laying in C. elegans by Salmonella lipopolysaccharides (LPS) is dependent on a Galpha protein expressed in chemosensory neurons"

Wilwayco, Kyla, Lee, Myeongwoo, Leung, Angela Ching-Yee

[International Worm Meeting, 2021]

Lipopolysaccharides (LPS) of Salmonella enterica serotype typhimurium stimulates egg laying in C. elegansN2 strain. Among the 23 G protein alpha subunits in C. elegans, we found that GPA-11 is a component in the signaling pathway, since the loss-of-function mutant did not respond to LPS stimulation while displaying wildtype stimulated egg-laying response to serotonin. We dissected the mutant hermaphrodites at the adult stage which are usually selected for egg-laying assays and found that they bore similar number of eggs to that of N2 worms. This confirms that the blunted response to LPS egg-laying stimulation in the mutants was not due to any limited number of eggs stored in the uterus, but was because of the absence of functional GPA-11 protein. The mutant was known to respond poorly to the repellent octanol but are wild-type for other traits studied. Therefore, our reported observation reveals another defect of the mutant. Besides, the defective egg-laying response to the bacterial toxin LPS is likely to be linked to innate immunity, so the involvement of the chemosensation-related GPA-11 that is expressed in the chemosensory ASHs and ADL neurons suggests that there is an interaction between chemosensation and innate immune responses. Moreover, GPA-11 is epistatic to TPH-1 involved in serotonin biosynthesis, but the G protein-coupled receptor with which GPA-11 interacts is yet to be identified. The egg-laying system responsive to LPS stimulation may provide insights into this complete signaling pathway. It is also noteworthy that GPA-11 in another Caenorhabditis species, C. briggsae, is dysfunctional because of an altered translation frame resulted from an insertion in the first exon. Future investigation is needed for understanding the possible dispensability of GPA-11 in innate immunity and/or chemosensation in the genus.

Tina Lai et al. (2001) International C. elegans Meeting "VAB-8 AND UNC-51 PROTEIN INTERACTIONS MEDIATE AXON OUTGROWTH"

Tina Lai, Gian Garriga

[International C. elegans Meeting, 2001]

In C. elegans most posteriorly directed cell and growth cone migrations require vab-8 , a gene that encodes at least two protein products known as VAB-8L and VAB-8S. VAB-8L is a 1066 amino acid protein that contains an N-terminal kinesin-like motor domain and functions in vab-8 -dependent growth cone migrations. VAB-8S is colinear with the C-terminal half of VAB-8L, and lacks the kinesin-like motor domain. VAB-8S is necessary for certain vab-8 -dependent cell migrations. To identify VAB-8-interacting proteins, we conducted a yeast two-hybrid screen using full length VAB-8L as bait. One protein identified was UNC-51, a serine/threonine kinase required for proper axon outgrowth (Ogura et al., 1994). UNC-51 was found to interact with another novel protein, UNC-14 (Ogura et al. 1997). We have found that both VAB-8 and UNC-14 bind the C-terminal 100 amino acids of UNC-51. Several observations suggest that VAB-8, UNC-14 and UNC-51 also interact in C. elegans . First, vab-8, unc-14 and unc-51 mutants display axon outgrowth defects. Second, all three genes are expressed in neurons that require vab-8 function. We have determined that vab-8 and unc-51 act cell autonomously for CAN axon migrations. Finally, misexpression of the UNC-51-binding domain of VAB-8L under control of the ceh-23 promoter results in a highly penetrant CAN posterior growth cone migration defect, a vab-8 phenotype, presumably by interfering with UNC-51 binding with wildtype VAB-8L. Misexpression of the VAB-8-binding domain of UNC-51 also produced CAN axon outgrowth defects, however, the defects extended to both anterior and posterior migrations. We are in the process of determining whether the misexpression phenotype can be suppressed by simultaneous misexpression of both protein fragments. The protein interactions and similar mutant phenotypes suggest that VAB-8 and UNC-51 act in the same pathway. Overexpression of vab-8 suppressed the posterior axon outgrowth defects of an unc-51 mutant, indicating a positive regulatory relationship between the two proteins. Overexpression of unc-51 in the CAN caused its axons to terminate prematurely (the loss-of-function phenotype) and to branch (a novel phenotype). We will test whether vab-8 mutations can suppress this branching phenotype. Suppression would suggest that vab-8 acts downstream of unc-51 . We are also taking biochemical approaches to determine the order of VAB-8 and UNC-51 action. We are testing whether UNC-51 can phosphorylate VAB-8 or whether VAB-8 can regulate UNC-51 autophosphorylation. Ogura et al. (1994) Genes & Dev. 8: 2389-2400. Ogura et al. (1997) Genes & Dev. 11: 1801-1811.